Glyoxalase I and formaldehyde dehydrogenase (FDH) are both glutathione (GSH)-dependent enzymes that catalyze the conversion of various aldehydes to thioester adducts of glutathione. Both enzymes must operate on a mixture of interconverting diasteriomeric hemimercaptals, formed in a preequilibrium step between the sulfur atom of glutathione and the carbonyl group of the aldehyde. That glyoxalase I can nonstereospecifically convert both diasteriomers directly to product is an outcome of a novel isotope-trapping method developed in this laboratory. The overall objective of the proposed research is to determine the catalytic significance and molecular basis of this observation as well as to test whether formaldehyde dehydrogenase has similar properties in common with glyoxalase I. To achieve this objective the following experiments are proposed: First, the substrate stereospecificity of FDH will be established by isotope trapping methods. Second, to evaluate the hypothesis that the "catalytic advantage" of nonstereospecific substrate binding is that enzymic catalysis is not limited by the nonenzymic rates of interconversion of the hemimercaptal diasteriomers, these nonenzymic rates will be determined, as a function of conditions, by nmr-broadening methods. Third, as a test that glyoxalase I and FDH catalyze the ipimerization of the bound diasteriomers as a parital reaction, the enzyme induced nmr line-broadening of the methine proton resonances of the diasteriomers will be tested for. Finally, the hypothesis that the ability of glyoxalase I to accomodate both diasteriomers as substrates is due to positional mobility of the glutathionyl sulfur atom of the bound diasteriomers will be indirectly evaluated from the binding conformation of isomeric substrate analogs to Mn++ glyoxalase I, as determined from the Mn++ to proton distances measured by paramagnetic effects on T1.